Enteropathogenic E. coli (EPEC) is a significant cause of infant diarrhea in developing countries. The hallmark of EPEC infection is their ability to cause attaching and effacing (AE) intestinal lesions, and all genes necessary for this phenotype are found within a 35.6 kb pathogenicity island termed the locus of enterocyte effacement (LEE), which encodes a type III secretion system. The LEE-encoded regulator Ler is critical for coordinate expression of gene products necessary for elaboration of the type III secretion system and the AE phenotype. [unreadable] [unreadable] The goal of this research is to elucidate the molecular mechanism of Ler-mediated transcriptional activation of EPEC virulence factors. During this period of support we will perform experiments to characterize the functional domains of Ler and investigate the "Ler-like" molecules of other Gram-negative pathogens, comparing their activities and DNA binding characteristics to those of Ler. We will correlate regulatory observations with the ability of EPEC to cause AE lesions on human epithelial cells in culture. EPEC and E. coli serotype O157:H7, a member of the enterohemorrhagic E. coli (EHEC) category of E. coli, are related pathogens. Like EPEC, EHEC causes AE intestinal lesions and possesses a LEE-encoded type III secretion system, including Ler. In the U.S., EHEC is of particular concern in food safety and public health because this organism has caused many outbreaks of bloody diarrhea due to contaminated meat products, produce and water. Perhaps even more urgently, diarrheagenenic E. coli, including EPEC and EHEC, are in the NAIAD Biodefense Research Priority Category B Pathogens. "Ler-like" molecules have been identified in a number of Gram-negative pathogens, and thus determining the molecular mechanism of Ler-mediated transcriptional activation in EPEC may lead to the development of effective therapies preventing disease caused by a wide range of pathogens. [unreadable] [unreadable]